First evidence of free-living Naegleria species in recreational lakes of Alberta, Canada

Canada's climate has changed and is projected to continue to do so over the coming decades. Annual average temperatures over the Canadian landmass have increased by 1.7 °C and the north has warmed on average by 2.3 °C since 1948 (Bush & Lemmen 2019).

Recently, western provinces of Canada recorded temperatures exceeding 40 °C with a new Canadian temperature record of 49.6 °C registered in the village of Lytton (World Weather Attribution 2021). The increase in average temperature across Canada could allow water temperatures to become more suitable for the growth of thermotolerant pathogens such as members of the free-living amoebae (FLA).

Amoebae belonging to the genera Naegleria are potentially pathogenic to humans and commonly found in warm freshwater environments such as lakes, groundwaters and thermal springs. Exposures (such as via forced nasal entry) with these amoebae may result in neurological infections. The best example is Naegleria fowleri, ‘the brain-eating amoeba’, the causal agent of primary amoebic meningoencephalitis (PAM), which is an acute and rapidly fatal infection of the central nervous system (CNS) (Marciano-Cabral 1988). Northern hemisphere evidence suggests that climate change may expand the natural habitat of N. fowleri further north (Gompf & Garcia 2019; Maciver et al. 2020; Gharpure et al. 2021).

To the best of our knowledge, no reports on Naegleria or other FLA species have been reported in the environmental water sources of Canada. As a scoping study, our aim was to evaluate the presence of Naegleria spp. in different recreational lakes across the province of Alberta, Canada.

Water samples were collected during the summer period from five different recreational lakes in the province of Alberta (Lac La Biche, Golden Sheaf Park, Lessard Lake, Wabamun Lake Provincial Park and Sylvan Lake) using 250-mL bottles (Fisherbrand™ 3001410008). Water samples were filtered using a vacuum manifold system with a 0.22-μm filter (MF-Millipore^® Millipore, USA). Filters were then cultured inverted onto 2% non-nutrient agar (NNA) plates supplemented with a thin film of viable Escherichia coli (ATCC25922) as a food source for the amoebae at 30 °C and were monitored daily under an inverted light microscope to detect the growth of amoebae.

The identification of Naegleria species was based on the morphological features of trophozoites, cysts and the flagellate transformation test (Pussard & Pons 1977; Page 1988; de Jonckheere 2006). The flagellate transformation was induced by suspending the trophozoites in 1 mL of distilled water at 37 °C for 30–60 min.

For a pure amoebae culture and further investigations, we established isolates that showed the typical Naegleria morphology in axenic cultures in Nunc™ 25-cm² tissue culture flasks (ThermoFisher Scientific, Canada) containing 5 mL of Serum Casein Glucose Yeast Extract Medium (SCGYEM) ATCC medium 1021 at 37 °C with 10% of fetal calf serum (de Jonckheere & van de Voorde 1977).

Amoebae were harvested from culture flasks and DNA extraction was performed by using the DNeasy Blood & Tissue Kit™ (Qiagen, Canada). Two sets of primers were used for the identification of Naegleria: (i) Naegleria genus-specific (5′-GAACCTGCGTAGGGATCATTT-3′ and 5′-TTTCTTTTCCTCCCCTTATTA-3′) designed from the rDNA ITS regions as described previously (Pélandakis et al. 2000), (ii) N. fowleri species-specific (5′-TTCCGAACCCACTCAATAAA-3′ and 5′-TTCCGAACCCTTAAAACCTC-3′) targeting a single-copy DNA region (Régoudis & Pélandakis 2016).

Reaction conditions for both sets were: 5 min at 94 °C, followed by 40 cycles at 94 °C for 1 min, 55 °C for 30 s, 72 °C for 2 min, and a final extension for 10 min at 72 °C. The resulting PCR products were purified using the QIAquick PCR purification kit™ (Qiagen, Canada) and sent to The Applied Genomics Core (University of Alberta) for sequencing and species identification based on sequence homology analysis by comparison to the available amoebae species sequences in the Genbank database.

Axenic cultures of amoebae were fixed at room temperature with 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences 15960). Samples were submitted for processing at the imaging core at University of Alberta, faculty of biological sciences.

Sectioned and carbon-coated samples were observed with a Hitachi H-7650 transmission electron microscope (TEM).

Despite the absence of N. fowleri, all four identified Naeglerial isolates are thermotolerant (Kilvington & White 1985), suggesting that the environments examined could promote the proliferation of N. fowleri. For example, recent cases associated with N. fowleri have been reported in Minnesota (USA), which shares a 547-mile border with the Canadian provinces of Manitoba and Ontario (Gompf & Garcia 2019). It is therefore not surprising that pathogenic Naegleria species could be present in Canada due to increasing water temperatures driven by climate change.

Moreover, food sources are known to play an important role in Naegleria spp. growth and presence (Goudot et al. 2012; Morgan et al. 2016; Puzon et al. 2020). As bacterivores, previous studies have shown that amoebae including Naegleria spp. uses a range of bacteria, especially Meiothermus spp. and E. coli as the preferred prey (Kyle & Noblet 1985; Schuster 2002; Xue et al. 2018; Puzon et al. 2020). In fact, environmental growth of E. coli has been reported in recreational Great Lakes (Verhougstraete et al. 2010) and faecal contamination related to increasingly intense storm events is another consideration for increased E. coli levels (Whitman & Nevers 2008).

Additionally, the isolated amoebae are known hosts of a temperature-selected pathogen Legionella pneumophila; a waterborne bacterium responsible for Legionnaires’ disease in engineered environments, soils and organic-rich media (Barbaree et al. 1986; Huang & Hsu 2010; Zbikowska et al. 2013; Dupuy et al. 2016). Therefore, increased FLA presence in water sources represents a potential risk, enhanced through the carriage of amoeba-resisting pathogens such as L. pneumophila (Thomas et al. 2010). To the best of our knowledge, this is the first report on the occurrence of Naegleria species in Canadian recreational waters, consistent with the worldwide distribution of Naegleria spp. in water sources (Saberi et al. 2020), and adding further information on the identification of Naegleria species in North America. As surface water temperatures continue to rise with climate change and the availability of bacterial food source increases, further investigations should be conducted to better understand the distribution and microbial ecology of problematic Naegleria spp. for water quality management strategies to prevent future amoebic infections.

These studies were supported by Alberta Innovates (grant # 201300490), Alberta, and Canadian Institute for Health Research (CIHR grant TGEHIPR 150713), Canada. We would like to thank Arlene Oatway for her help with transmission electron microscopy (Microscopy Facility Biological Sciences, University of Alberta).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.